Investigation of the gastroprotective activity of some drugs in indomethacin-induced gastric lesion in rats

Gastric ulcer NSAIDs Sildenafil Verapamil, Propranolol Abstract Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) increases the risk of severe gastric events. To minimize these risks, patients often require concomitant acid-suppressive therapy. This study was conducted to investigate the putative gastroprotective effect of sildenafil, verapamil and propranolol and possible mechanisms underlying the effect of these drugs in experimentally-induced gastric lesions in rats. Rats were assigned to vehicle (saline), control (indomethacin, 30-mg/kg, p.o.), sildenafil (10-mg/kg, p.o.), verapamil (10 mg/kg, p.o.), propranolol (10 mg/kg, p.o.) and ranitidine (50-mg/kg, p.o); the drugs were administered 30-minute prior to indomethacin. After 4-hours, all rats were sacrificed, and stomach of each rat examined for gastric lesions either for biochemical or histopathological analysis. Indomethacin induced marked ulcerative lesions in form of strikes in the gastric mucosa. Furthermore, pre-treatment with sildenafil, verapamil, and propranolol significantly reduced gastric acid secretion, ulcer scores, and lipid peroxides. Moreover, they markedly protect the stomach against indomethacin effect. The results confirm that each drug has a gastroprotective effect but with different mechanisms in prevention.


Introduction
Peptic ulcers are multi-etiologic, frequently recurrent and widespread chronic disease (Süleyman et al., 2001).Peptic ulcers occur mainly in the stomach (gastric ulcer) or proximal duodenum (duodenal ulcer), and they continue to be a common disease that causes a substantial socioeconomic burden and negatively impacts on quality of life (Lee et al., 2010).
The pathogenesis of gastric ulcers is based on complex interactions between aggressive and protective factors (Prabha et al., 2009).Nonsteroidal anti-inflammatory drugs (NSAID) are known to be aggressive agents for gastric ulcer development (Kamel et al., 2009), they follow H. pylori in the ulcer etiopathogenesis (Bulent et al., 2007).As the prevalence of H. pylori, the infection has declined in worldwide; gastric ulcer has become more commonly associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) (Makola et al., 2007).Indomethacin causes damage in the gastric mucosa and impairs ulcer healing as an adverse effect.Although the inhibition of cyclooxygenase (COX), which leads to depletion of endogenous prostaglandins (PGs), is a major pathogenic factor, it is unlikely that PG deficiency alone is sufficient to initiate the process that ultimately results in gastric ulceration (Takeuchi et al., 2001).
There are many anti-ulcerogenic drugs, among which the most efficient is omeprazole.However, these drugs do not always provide an effective treatment of ulcer (Bulent et al., 2007).Therefore, treatment of ulcers is still a major problem, and new drugs are urgently needed for the treatment of gastro-duodenal ulcer.
Sildenafil (SLD) is currently used in the treatment of functional impotence; it increases the efficiency of the guanosine cyclic 3', 5'-monophosphate (cGMP),

Induction of Experimental gastric lesion
Rats were fasted overnight before starting the experiments.Rats received an acute dose of indomethacin (30 mg/kg, p.o.).The indomethacintreated rats were examined for gastric lesions four hours after indomethacin administration.

Anesthesia:
After four hours, rats were anesthetized in a jar with a tight-fitting lid containing an appropriate amount of ether and sacrificed by cervical dislocation.After that, a laparotomy was performed and the stomach of each rat was dissected after pyloric ligation.

Acid determination
Gastric secretion was determined by the method of Aguwa et al., (1984).The gastric contents were collected by washing with 1 ml of saline and centrifuged at 3000 rpm for 10 min.The total acid concentration was determined in the supernatant by titration to pH 7.0 with 0.01N NaOH using phenolphthalein as indicator.After that, pH value of the gastric juice was calculated by the following formula: pH = -log [H +]

Quantification of ulceration
The glandular portion comprising of the fundic and corpus region of each stomach was opened longitudinally along the greater curvature and examined microscopically.The number and severity of lesions in the glandular mucosa were scored from 0 to 5 according to the method of Clement et al., (1998).

0
No lesion 0.5 diffuse hyperemia 1 1 to 2 small ulcers 1.5 3 to 6 small ulcers 2 7 to 10 small ulcers 2.5 More than 10 small ulcers which displays an inhibitory effect on the smooth muscle cells of the arterioles.Accumulating evidence from both animal and human studies indicates that NO plays critical roles in normal wound repair.The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis and inflammation (Pan et al., 2005).
Verapamil is a widely used for clinical treatment of cardiovascular diseases.It is accepted that calcium antagonists inhibit mobilization of calcium from intracellular stores with resultant inhibition of calcium effects on gastric function (Candido et al., 1993).
In the present study, we aimed to examine the putative gastroprotective effect of SLD, verapamil, and propranolol in indomethacin-induced gastric mucosal lesions.Further, the present study was extended to investigate possible mechanisms underlying their gastroprotective effect.

Animals
Sixty male albino rats weighing 150-200 g were used in the present study.Rats were housed in stainless steel cages with free access to food and water under controlled laboratory condition (temperature 25±3 and regular dark-light cycle).Rats were habituated to the experiment condition 10 days before conduction of the experiment.
All experiments were done under approval from institutional animal use & care committee.

Drugs
Indomethacin

Preparation of tissue homogenate
0.5 g of stomach tissues were homogenized in phosphate-buffered saline (PBS) pH 7.4 using a glass-Teflon homogenizing tube (Glas Col homogenizer system, Vernon hills, USA).The homogenate was centrifuged at 2500 rpm for 10 minutes, and the supernatant was carefully removed from the pellet and used for biochemical analyses.

Determination of nitric oxide in tissues
Nitrite was determined as an oxidation product and indicator of NO synthesis as described previously by Moshage et al., (1995).
The method is based on the addition of Griess reagent which converts nitrite into deep purple azo chromophore.No levels was expressed as µmol/g tissue.

Determination of lipid peroxides
Lipid peroxides (LPs) were assessed according to the method of Ohkawa et al. ( 40) based on the reaction with thiobarbituric acid using 1,1,3,3, tetra methoxy propane as a standard.

Estimation of GSH concentration in tissue:
3 1 marked ulcer plus 0 to 4 small ulcers 3.5 1 marked ulcer plus 5 or more small ulcers 4 2 marked ulcers plus 0 to 4 small ulcers 4.5 2 marked ulcers plus 5 or more small ulcers 5 3 or more marked ulcers Then, the protective effect of the drugs was evaluated by calculating % inhibition of ulceration by the following formula: inhibition of ulceration = [ (ulcer index in control -ulcer index in the test) / ulcer index in control] X 100 (Yinusa et al., 2000).

Mucin assay
Following a reported method, free mucin in the gastric tissue was estimated.Briefly, 0.5 g of the glandular segment of the stomach was added to 10 ml of 0.1% alcian blue solution (in 0.16 M sucrose buffered with 0.05 M sodium acetate; final pH was adjusted to 5.5).
The stomach tissue was left to be stained for 2 hrs in the alcian blue solution.After that, the uncomplexed dye was removed by two washes with 0.25 M sucrose.The Comlexed dye was then eluted by immersion in 5 ml of 0.5 M magnesium chloride for 2 hrs.Dye extract was shaken briefly with an equal volume of diethyl ether and then centrifuged at 3600 rpm for 10 minutes.The optical density of the aqueous layer was read at 598 nm.A standard curve of alcian blue was plotted using different concentrations of alcian blue  for the statistical analysis.For all comparisons, differences were considered significant at p≤0.05.

Results and Discussion
In the present study, oral administration of indomethacin (30 mg/kg, p.o.) induced a significant increase in the total acid concentration (3.38 ± 0.16 g/L) as compared with the saline group (1.3 ±0.12 g/L, P ≤ 0.05, Table 3).Accordingly, the pH value of the gastric juice in indomethacin group was significantly lower than saline group (1.01 ± 0.03 vs. 1.45 ± 0.05, p≤ 0.05, Table 1, Figure 1).
Additionally, indomethacin treatment induced a marked increase in the ulcer score as compared to saline group (5 ± 0 vs. 0).Meanwhile, the percent of inhibition was significantly lower than the saline group (0% vs. 100%, p≤ 0.05, Table 2, Figure 2).
Pretreatment with Ranitidine suppressed the acid production and subsequently elevated the pH value as compared to indomethacin group (0.57±0.06 and 1.85±0.05vs. 3.38±0.16and 1±0.03, respectively, p≤0.05,Table 3).Furthermore, Ranitidine attenuated the ulcer score and increased % inhibition of ulceration as compared to indomethacin group.
Similar to ranitidine, pretreatment with Sildenafil, Verapamil or Propranolol markedly suppressed acid production and increased pH value as compared to indomethacin group (p≤ 0.05, Table 3).
Glutathione in the stomach homogenate was measured according to the colorimetric method of Sedlak and Lindsay (1968).The glutathione level in the gastric mucosa was expressed as nano mole per milligram tissue.

Determination of TNF-α
TNF-α was determined according to Mysliwska et al.

Histopathological examination
For histological assessment, the glandular area of stomach was fixed in 10% phosphate-buffered paraformaldehyde solution and prepared for staining with, hematoxylin and eosin and then examined under a light microscope.The specimens were assessed according to the criteria of Laine and Weinstein (1988).In brief, a 1 cm length of each histological section was assessed for epithelial cell loss ( a score of 0-3), edema in the upper mucosa ( a score of 0-4), hemorrhagic damage ( a score of 0-4) and presence of inflammatory cells ( a score of 0-3).The sections the sections were assessed by an experienced pathologist without the knowledge of treatments.

Statistical Analysis
Data were expressed as mean±SEM and statistically analyzed using SPSS program version 16.Oneway analysis of variance, ANOVA, followed by Bonferroni's multiple comparisons test are used with the exception of NO.Mucin and GSH were significantly decreased.However, LPs and TNF-α were significantly elevated in the stomach tissue of indomethacin group (p≤0.05, Figure 3).
Pretreatment with Ranitidine significantly restored tissue mucin concentration and suppressed the production of LPs in the stomach as compared to indomethacin-group.Ranitidine group showed higher TNF-α level as compared to Indomethacin-group (p≤0.05, Figure 3).Pretreatment with Sildenafil Moreover, the mean ulcer score was attenuated by pretreatment with these drugs as compared to indomethacin group (p≤ 0.05, Table 3).
However, the inhibition % of ulceration was significantly enhanced by these drugs as compared to indomethacin group (91%, 80.6%, 60% vs. 0%, respectively, p≤0.05,Table 3).On the other hand, the biochemical parameters measured in the stomach tissue differed significantly in Indomethacin-treated rats as compared with saline-treated rats; this Mucin and LPs suppress production in the stomach tissue as compared to indomethacin group (p≤0.05,Table 5).
From the histological point of view, Administration of indomethacin (30 mg/kg, p.o.) induced marked erosion to the mucosal epithelia coupled with congestion and bleeding.Also, inflammation was unexpectedly decreased the LPs production as compared to indomethacin group (p≤0.05, Figure 3).
Furthermore, Verapamil could enhance Mucin and GSH and suppress LPs production as compared to Indomethacin-group (p≤ 0.05, Figure 3).
Finally, pretreatment with Propranolol could elevate

Figure 1 :
Figure 1: Total acidity (panel-A) and pH value (panel-B) in the gastric juice of indomethacin-treated rats (30 mg/kg, p.o.) and the effect of pretreatment with different drugs.

Figure 2 :
Figure 2: Ulcer scores (panel-A) and % inhibition of ulceration (panel-B) in the gastric juice of indomethacin-treated rats (30 mg/kg, p.o.) and the effect of pretreatment with different drugs.Administration of indomethacin (30 mg/kg, p.o.) induced a significant increase in the ulcer score as compared to saline group (p≤0.05).Pretreatment with ranitidine (50 mg/kg, p.o.), SILD (10 mg/kg, p.o.), verapamil (10 mg/kg, p.o.) or propranolol (10 mg/kg, p.o.) suppressed ulcer score and improved the % inhibition of ulceration as compared to indomethacin group (p≤0.05).% inhibition = [(ulcer index in control -ulcer index in test) / ulcer index in control] X 100.Data are expressed as mean±SEM and analyzed using ANOVA followed by Bonfferroni's multiple comparisons test (n=8).#: significantly different from saline group at p≤0.05.*: significantly different from indomethacin group at p≤0.05.*: significantly different from indomethacin group at p≤0.05.

Figure 3 :
Figure 3: The antioxidant and anti-inflammatory action of the different drugs in the gastric juice of indomethacin-treated rats (30 mg/kg, p.o.).